The novel pyrrolo-1,5-benzoxazepine, PBOX-15, synergistically enhances the apoptotic efficacy of imatinib in gastrointestinal stromal tumours; suggested mechanism of action of PBOX-15
The C-KIT receptor tyrosine kinase is constitutively activated in the majority of gastrointestinal stromal tumours (GIST). Imatinib (IM) a selective inhibitor of C-KIT, is indicated for the treatment of KIT-positive unresectable and/or metastatic GIST, and has tripled the survival time of patients with metastatic GIST. However, the majority of patients develop IM-resistance and progress. Although IM elicits strong antiproliferative effects, it fails to induce sufficient levels of apoptosis; acquired IM-resistance and disease recurrence remain an issue, a more effective drug treatment is greatly needed. We examined the effect of a novel microtubule-targeting agent (MTA), pyrrolo-1,5-benzoxazepine (PBOX)-15 in combination with IM on GIST cells. PBOX-15 decreased viability and in combination with IM synergistically enhanced apoptosis in both IM-sensitive and IM-resistant GIST cells, decreased the anti-apoptotic protein Mcl-1, and enhanced activation of pro-caspase-3 and PARP cleavage. The combination treatment also led to an enhanced inhibition of C-KIT-phosphorylation and inactivation of C-KIT-dependent signalling in comparison to either drug alone; CDC37, a key regulator of C-KIT in GIST was also dramatically decreased. Furthermore, PBOX-15 reduced CKII expression, an enzyme which regulates the expression of CDC37. In conclusion, our findings indicate the potential of PBOX-15 to improve the apoptotic response of IM in GIST cells and provide a more effective treatment option for GIST patients.
KeywordsGastrointestinal Imatinib Microtubules CKIT CDC37 CKII
Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumour of the gastrointestinal tract and are characterised by activating mutations, usually in exon-9 or −11, of the stem cell receptor tyrosine kinase (C-KIT) (85 %) or the platelet-derived growth factor receptor A kinase (PDGFRA) (7 %) [1, 2]. Targeted therapy with the tyrosine kinase inhibitor imatinib mesylate (IM; Glivec®, Novartis Pharmaceuticals), which inhibits KIT and PDGFRA activity is a highly effective treatment for metastatic GIST. Prior to the use of IM, recurrent or metastatic GIST was uniformly fatal. IM was originally developed as an inhibitor of the ATP binding site of the breakpoint cluster region-abelson (BCR-ABL) fusion onco-protein, which plays a central role in the oncogenesis of chronic myelogenous leukaemia (CML) . IM also targets the ATP binding site of KIT, causing inhibition of autophosphorylation, and disruption of the downstream signalling pathways involved in cellular proliferation and survival such as mitogen-activated protein (MAP) kinase and AKT signalling pathways .
Although >85 % of patients with metastatic GIST benefit from IM therapy, complete responses are rare and the majority of patients develop resistance to IM during the course of treatment. It is critical to identify novel strategies or new agents that induce GIST cell apoptosis either as single agents or in combination with IM.
Recently we identified a series of novel MTAs, the PBOX compounds, which behave as tubulin depolymerisers and possess the ability to potently induce apoptosis in several cancerous cell lines [5, 6, 7]. The PBOXs also induce apoptosis in ex vivo chronic lymphocytic leukaemia  and CML patient samples including those that are resistant to IM . We have shown that PBOXs impair the growth of tumours in vivo in breast cancer and CML mouse tumour models. As GIST and CML are both cancers characterised by a unique oncogenic dependency on a tyrosine kinase we sought to evaluate the therapeutic potential of PBOX-15 in GIST. Collectively these results suggest the PBOXs are a promising group of potential anti-cancer therapeutics. We decided to examine the potential of combining IM with PBOX-15 as a novel treatment option for GIST. We hoped to decrease cell viability and increase the apoptosis in GIST cells by targeting both C-KIT with IM and microtubules with PBOX-15.
Materials and methods
GIST-T1 was developed by Takahiro Taguchi (Kochi University, Kochi, Japan) an IM-sensitive metastatic GIST and GIST-T1-Juke is an IM-resistant subline that has a secondary KIT-resistance mutation D816E . GIST-T1 cells cultured in DMEM/F12 + GlutaMAX medium supplemented with 10 % (v/v) foetal bovine serum (FBS) and 1 % (v/v) penicillin/streptomycin. GIST-T1-Juke cells grown in RPMI 1640 + GlutaMAX medium supplemented with 15 % (v/v) (FBS) and 1 % (v/v) penicillin/streptomycin. Cells were incubated in a humidified environment at 95 % O2 and 5 % CO2, passaged twice a week.
Pyrrolo-1,5-benzoxazepine-15 (PBOX-15) was synthesised as described previously  dissolved in ethanol and stored at -20 °C. Imatinib (Novartis) was reconstituted in DMSO (10 mM stock) and stored at -20 °C. Media sourced from Biosciences, and FBS obtained from Gemini Bioproducts. The BCA reagents from Pierce (Illinois, U.S.), the polyvinylidene difluoride membranes from Millipore (Cork, Ireland), protease inhibitors from Roche (Clare, Ireland). Chemicals from Sigma-Aldrich (Sigma Aldrich, St Louis, MO, USA), cell culture materials from Greiner Bio-One GmbH (Stonehouse, U.K.).
AlamarBlue monolayer viability assay
Cells were seeded at densities varying from 4 000–5 000 cells/well, 96-well plate, 200 μL medium, left overnight to attach, treated with a range of concentrations of PBOX-15 and IM for 24 or 48 h. AlamarBlue (final concentration 10 % (v/v)) added to each well and left to incubate in the dark at 37 °C for 5 h. Fluorescence was measured using a SpectraMax Gemini plate reader (Molecular Devices, Sunnyvale, CA) at excitation and emission wavelengths of 544 nm and 590 nm. The mean of each triplicate was calculated. Vehicle treated wells were set at 100 % viability and treated wells were calculated as a percentage of the vehicle control. Dose response curves were plotted and IC50 values obtained using Prism GraphPad 4.
Following treatment, cells were harvested and fixed in 70 % ethanol/PBS. Fixed samples were stored at -20 °C until required. 4 mLs of PBS was added to each sample, ethanol was removed by centrifugation, pellets were incubated in 400 μL FACS flow sheath fluid supplemented with 10 mg/mL RNase A (Sigma Aldrich,
St Louis, MO, USA) and 100 mg/mL propidium iodide (PI) (Sigma Aldrich, St Louis, MO, USA). Cells were incubated in the dark at 37 °C for 30 mins. Analysis was performed on a FACScalibur Fluorescence Associated Cell Sorter (FACS) (Becton–Dickinson, San Jose, CA, USA) using Cell Quest and Quanti-Quest software. Samples were gated using a vehicle control to eliminate debris and cell aggregates from analysis. 10,000 cells from each sample were counted and results visualised on histrograms.
Analysis of drug interactions
Using the software programme Calcusyn, median dose analysis was undertaken to study interactions between different drugs. This method is based on the drug effect equation of Chao and Talalay  providing quantitative determination for synergism. The lower the combination index value (CI), the greater the degree of synergism (CI < 1), additive effect (CI = 1) and antagonism (CI > 1). A CI value of 0.1–0.3 indicates strong synergism, 0.3–0.7 indicates synergism, 0.7–0.85 indicates moderate synergism and 0.85–0.9 indicates slight synergism.
Statistical analysis of experimental data was performed using the computer program Prism Graph Pad 4. Results were presented as mean ± S.E.M. For comparison of two groups, values were determined using a Student’s paired t test. A value of P < 0.05 was deemed to be significant.
Cells were harvested in Ripa buffer (R0278, Sigma-Aldrich); DTT (to a final concentration of 50 mM), 0.313 M Tris HCL pH 6.8, 10 % SDS and 0.25 % bromophenol blue, 50 % glycerol were added to each sample followed by boiling for 4 mins at 95 °C. Equal quantities of protein (as determined by a BCA assay) were separated on polyacrylamide gels followed by transfer to PVDF membranes. Membranes were blocked in 5 % blocking serum (Bio-Rad) in PBS-tween (0.1 %) for 1 h. Membranes were incubated in the relevant primary antibodies overnight, washed and incubated in secondary antibody for 1 h and washed again. Enhanced chemiluminescence (Millipore, Cork, Ireland) was used for detection of protein expression. Western blot analysis was performed using antibodies directed against PARP (AM30, Millipore), Pro-Caspase-3 (Merck Biosciences), Mcl-1 (Am50, Calbiochem), P-C-KIT (Tyr719, Cell Signalling (CS), C-KIT (Ab81, CS), P-Akt (S473, CS), Akt (C67E7, CS), P-PTEN (S380, CS), P-CDC37 (Ser13, CS), CDC37 (D28H7, CS), HSP90 (610,418, BD Biosciences), CKIIα (A300-196 A-T, Bethyl), (with incubation with a horseradish peroxidase-conjugated anti-mouse, anti-rabbit antibody (Promega, Madison, WI, USA)). Blots were probed with β-actin to confirm equal loading. 48 h β-actin is shown and is representative of equal loading of all blots.
Imatinib and PBOX-15 reduce the viability of GIST cells in an AlmarBlue monolayer viability assay
PBOX-15 synergistically enhances the apoptotic effect of imatinib in both sensitive and resistant GIST cells via flow cytometric analysis
PBOX-15/imatinib combination synergistically enhance apoptosis in GIST-T1 (imatinib-sensitive GIST cells)
% Sub G1/G0 Cells
% Sub G1/G0 Cells
PBOX-15/imatinib combination synergistically enhance apoptosis in GIST-T1-Juke (imatinib-resistant GIST cells)
% Sub G1/G0 Cells
% Sub G1/G0 Cells
Western blot analysis of pro-caspase-3 and of poly (ADP) ribose polymerase (PARP) cleavage in GIST cells confirmed the sub G0/G1 peaks observed by flow cytometric analysis were representative of an apoptotic cell population. Consistent with results from flow cytometry, the combination caused the disappearance of the 32 kDa band of pro-caspase-3 indicating its activation (Fig. 2b). Enhanced PARP cleavage was observed with the combination in both GIST-T1 and Juke cells in comparison to either agent alone.
PBOX-15 treatment is accompanied by an enhanced downregulation of anti-apoptotic mcl-1 in GIST-T1 and GIST-T1-juke cells
PBOX-15/imatinib combination inhibits KIT and KIT-Dependent signalling pathways in both imatinib-sensitive and imatinib-resistant GIST cells
To elucidate the mechanism of PBOX-15 induced sensitisation to apoptosis, we investigated the effects of PBOX-15 on KIT and KIT-dependent signalling pathways, alone and in combination with IM. Both IM and PBOX-15 suppressed C-KIT phosphorylation and total C-KIT expression in IM-sensitive GIST-T1 cells; the combination treatment gave enhanced inhibition of P-C-KIT at 24 and 48 h. This result was paralleled by a substantial inhibition of the downstream PI3K/Akt pathway as monitored by the suppression of both P-Akt at 24 h and total Akt at 48 h.
Imatinib and PBOX-15 supresses CDC37 in both imatinib sensitive and resistant GIST
There is an urgent need for apoptosis inducing agents that can be administered in combination with IM. Studies have shown the use of a MTA in combination with IM to be very promising [18, 19]. We demonstrate for the first time the potential benefit of targeting KIT with IM while inhibiting the assembly of tubulin with the MTA, PBOX-15. We have shown IM combined with PBOX-15, synergistically enhances apoptosis in both IM-sensitive and IM-resistant GIST cells: 52 % increase in apoptosis in IM-sensitive cells and 34 % apoptosis in IM-resistant GIST cells. IM has previously been reported to induce cell cycle arrest at G0/G1 in GIST cells  whilst PBOX compounds induce G2M arrest , this correlates with this study, the targeting of the cell cycle at these two phases, may have resulted in the synergistic apoptotic effect seen. Treatment with the combination of IM and PBOX-15 resulted in enhanced activation of pro-caspase-3 and PARP cleavage in comparison to either agent alone which correlates with the flow cytometric data.
To further confirm the enhanced apoptotic effect of PBOX-15 and IM in our GIST cells we examined the expression of an anti-apoptotic protein Mcl-1 following treatment. Mcl-1 has been purported to be a critical regulator of apoptosis induced by MTAs [16, 21]. As PBOX-15 is a MTA we postulated that it may affect expression of Mcl-1 in GIST cells. Mcl-1 was strongly down-regulated by PBOX-15 in both IM-sensitive and –resistant GIST cells, consistent with previous PBOX studies [5, 16].
As GIST is highly dependent on the PI3K-Akt pathway for survival, in particular C-KIT expression, we examined the effect of the drug treatments on this pathway. PBOX-15 combined with IM had a much stronger inhibitory effect than IM alone on P-C-KIT in both IM-resistant and IM-sensitive GIST cell lines, this correlated with a strong decrease in Akt showing an overall reduction in the activation of the PI3K/Akt pathway. Interestingly, the IM-resistant GIST-T1-Juke cells exhibited no decrease in P-C-KIT when treated with a high concentration of IM alone; however, PBOX-15 gave a strong decrease in P-C-KIT in these cells, which correlated with the effect seen on P-Akt. PBOX-15 appeared to overcome IM resistance. A negative regulator of the PI3K/Akt pathway, PTEN, was unaffected by either drug treatment, suggesting PTEN is not targeted. GIST-T1-Juke IM resistance is possibly not associated with loss of expression of PTEN as has been the case with other IM-sensitive GISTs .
KIT is regulated by the chaperone Heat Shock Protein 90 (HSP90) . Recently Marino-Enriquez et al.  have shown CDC37, a co-chaperone of HSP90, to be a more specific target of KIT . CDC37 associates with a large subset of HSP90 client proteins, primarily protein kinases  which are mainly involved in signal transduction, cell proliferation and survival. CDC37 interacts with KIT and regulates its expression, activation and downstream signalling targets in GIST . In addition a positive feedback loop between casein kinase 2 (CKII) and CDC37 promotes the activity of multiple protein kinases and in turn CDC37 protects CKII from inactivation . CKII is overexpressed in all cancers and is a key player in cell growth, survival and apoptosis, with over 300 substrates [26, 27, 28], its high expression in cancer cells is indicative of its importance in tumourigenesis.
We examined the expression of HSP90, CDC37, P-CDC37 and CKIIα (the active form of CKII ) in our cells after treatment. A down-regulation of HSP90 in IM-sensitive GIST cells was observed in response to IM alone, whereas in the IM-resistant GIST cells a decrease in HSP90 was elicited by PBOX-15. Both IM and PBOX-15 decreased P-CDC37 expression; however a stronger decrease was found with the combined treatment in both IM-sensitive and IM-resistant GIST cells, a similar effect to that observed with P-C-KIT. It is possible that in GIST-T1 PBOX-15 is independently inhibiting CDC37 function even though it does not appear to inhibit HSP90. In addition, PBOX compounds target microtubules and CDC37 regulates CKII, CKII binds to microtubules and has an important role in the maintenance of cell morphology . PBOX compounds have been shown previously to depolymerize microtubules by inhibiting the assembly of purified tubulin, indicating the molecular target of PBOX is in fact tubulin . Therefore, the PBOX disruption of the microtubules may prevent the binding of CKII to microtubules. To our knowledge this is the first time a MTA has been shown to inhibit CDC37 activity. A decrease in CDC37 possibly resulted in a downstream decrease in C-KIT, resulting from the loss of the regulatory feedback loop with CKII. This correlated well with a strongly reduced expression of CKIIα.
In conclusion, we have shown for the first time, the combined treatment of IM and PBOX-15 synergistically enhances apoptosis in both IM-sensitive and IM-resistant GIST cells, concomitant with a strong decrease in C-KIT, CDC37 and CKII, all of which play a significant role in maintaining the malignant phenotype of GIST. These findings indicate the potential of PBOX-15 to improve GIST treatment.
We would like to thank Novartis Pharma AG, Basel, Switzerland for their kind donation of imatinib. We would also like to thank Dr. Jonathan Fletcher for his initial advice with this project.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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